Exposure to drugs and toxic metals results in the acquisition of resistance mechanisms. Bacterial resistances are nearly all transcriptionally regulated. The overall goal of this study is to gain insights into the evolution and organization of novel metal binding motifs in the regulatory proteins that control expression of bacterial resistances. The clinically isolated resistance plasmids R773 and pl258 carry the arsenical resistance (ars) and cadmium resistance (cad) operons that encode ATP-coupled extrusion pumps for As(lll)/Sb(lll) and Pb(ll)/Cd(ll)/Zn(ll), respectively. The ArsR and CadC repressors are two small homologous metal binding proteins responsible for metalloregulation of gene expression of the ars and cad operons, respectively. In plasmid-encoded ars operons there is a second unrelated As(lll)/Sb(lll)-responsive repressor, ArsD. Recent evidence indicates that ArsD serves as a metallochaperone for the As(lll)- translocating ArsAB pump. Specific Aim 1. Structure and function of the S. aureus plasmid pl258 CadC: Two distinct types of metal sites, one within the DNA binding site and the other at the dimer interface, are observed in the crystal structure of CadC. The properties and function of each site will be explored by using a combination of molecular genetic, biochemical, biophysical and structural approaches. Specific Aim 2. Structure and function of ArsR As(lll)-responsive repressors: Two aspects of ArsR structure and function will be analyzed. First, conformational change induced by As(Ill) binding will be probed. Second, the evolution of As(Ill) binding sites will be explored. Specific Aim 3. Roles of ArsD as a metalloregulator and a metallochaperone: The properties of ArsD that allow it to function as a repressor of the ars o/p will be explored, as will its role as a metallochaperone for intracellular transport of As(Ill) to the ArsAB As(Ill) extrusion pump. The ars repressors and homologues provide valuable models for the study of the regulation of drug and metal resistances: we have the ability to combine classical bacterial genetics and modern molecular biology with biochemical, biophysical and structural approaches.